Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Endothelin-1 (ET-1) is secreted from endothelial and vascular smooth muscle cells (VSMC) after intracellular hydrolysis of big ET-1 by endothelin converting enzyme (ECE). The metallopeptidase called ECE-1 is widely thought to be the physiological ECE, but unequivocal evidence of this role has yet to be provided. Endothelial cells express four isoforms of ECE-1 (ECE-1a, ECE-1b, ECE-1c, and ECE-1d), but the identity of ECE-1 isoforms expressed in VSMC is less clear. Here, we describe the characterization of ECE-1 isoforms in bovine pulmonary artery smooth muscle cells (BPASMC) and the effect on ET-1 synthesis of an antisense oligodeoxynucleotide (ODN) to ECE-1c. Reverse transcriptase-polymerase chain reaction (RT-PCR) evaluation of total RNA from BPASMC showed that ECE-1a and ECE-1d were not expressed. Sequencing of cloned ECE-1 cDNA products and semiquantitative RT-PCR demonstrated that ECE-1b and ECE-1c were expressed in BPASMC, with ECE-1c being the predominant isoform. Basal release of ET-1 from BPASMC was low. Treatment for 24 h with tumor necrosis factor-alpha (TNFalpha) stimulated ET-1 production by up to 10-fold with parallel increases in levels of preproET-1 mRNA. Levels of ECE-1c mRNA were also raised after TNFalpha, whereas amounts of ECE-1b mRNA were decreased significantly. Treatment of BPASMC with a phosphorothioate antisense ODN to ECE-1c caused a marked reduction in ECE-1c mRNA levels and ECE-1 protein levels. However, basal and TNFalpha-stimulated ET-1 release were largely unaffected by the ECE-1c antisense ODN despite the inhibition of ECE-1c synthesis. Hence, an endopeptidase distinct from ECE-1 is mainly responsible big ET-1 processing in BPASMC.
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PMID:Effect of an antisense oligodeoxynucleotide to endothelin-converting enzyme-1c (ECE-1c) on ECE-1c mRNA, ECE-1 protein and endothelin-1 synthesis in bovine pulmonary artery smooth muscle cells. 1116 Aug 49

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metalloprotease responsible for production of vasoactive endothelin (ET)-1 from inactive big ET-1. ECE-1 exists as four separate isoforms, ECE-1a, b, c, and d, which differ only in their amino-terminal regions. We investigated the expression and localization of the ECE-1 isoforms in primary human umbilical vein endothelial cells (HUVECs) and EAhy926 cells. Reverse transcriptase polymerase chain reaction showed expression of all four isoforms in both cell lines, with ECE-1d seeming, at least qualitatively, to be the predominant isoenzyme. Isoform-specific polyclonal antibodies were used to investigate isoform protein expression. ECE-1a, b, and c protein was detected in EAhy926 cells by immunoblotting; only ECE-1a and ECE-1c were detected in HUVECs. Using immunofluorescence microscopy analysis, both HUVEC and EAhy926 cells showed nuclear immunoreactivity with a monoclonal antibody recognizing all ECE-1 isoforms. The ECE-1a antibody also showed nuclear immunoreactivity in both cell lines; this seemed to colocalize with nucleolin. The ECE-1b antibody showed nuclear immunoreactivity in EAhy926 cells, but no overlap with nucleolin was seen. Intracellular immunoreactivity was seen in both cell lines using the ECE-1c antibody; this showed some colocalization with concanavalin A (an endoplasmic reticulum marker). von Willebrand Factor was used as a marker for Weibel-Palade bodies in HUVECs, but no colocalization with ECE-1 was seen during this study. The data presented here sheds new light on the localization of ECE-1a, b, and c in cultured human endothelial cells, which may further understanding of the ET system and aid design of therapeutic ECE inhibitors.
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PMID:Expression and localization of endothelin-converting enzyme-1 isoforms in human endothelial cells. 1674 Sep 87